This invention relates to immunoassays, and more particularly to such assays wherein a fluorescent tag capable of emitting fluorescent radiation when excited by more energetic exciting radiation is incorporated into a constituent of an antigen-antibody or similar complex.
Immunoassays, in which aliquots of sample and one or more reagents are variously reacted to form antigen-antibody or similar complexes which may then be observed in order to assay the sample for the presence and titer of a predetermined moiety of the complex, are well known. Typical of such assays are those wherein a specific antibody is used to measure the quantity of the antigen for which it is specific (or vice versa). However, the technique has been extended to quantitate haptens (including hormones, alkaloids, steroids, and the like) as well as antigens, and antibody fragments (i.e., Fab) as well as complete antibodies, and it is in this broader sense that the present invention should be understood.
Sensitive immunoassays employ tracer techniques wherein a tagged constituent of the complex is incorporated into the reagent, the non-complexed tagged reagent being separated from the complexed reagent, and the complex (or non-complexed reagent) then quantitated by observing the tag. Both radioisotopes and fluorescent markers have been used to tag constituents of immunoassay reagents, the tag being respectively observed by a gamma ray counter of a fluorometer. The present invention is, however, directed only to those assays which rely on fluorescence.
The separation of the non-complexed tagged moiety from the complexed is commonly accomplished by immobilizing a predetermined one of the components of the complex to a solid phase (such as the inside wall of a test tube, glass or polymeric beads, or the like) in such a way as not to hinder the component's reactivity in forming the complex. As an example, an antibody such as immunoglobulin G (IgG) may be bound by its carboxyl terminations to a solid phase, such as glass, by a silyl compound such as 3-aminopropyltrimethoxysilane, thereby leaving the antibody's antigen reactive amino terminations free. Any complex formed incorporating the immobilized component may then be physically separated from the non-reacted complement remaining in solution, as by aspirating or decanting the fluid from a tube or eluting the fluid through a particulate bed.
In competitive immunoassay, the reagent consists of a known quantity of tagged complement (such as antigen) to the immobilized component of the complex (in this instance, antibody). The reagent is mixed with a fixed quantity of the sample containing the untagged complement to be quantitated. Both tagged and untagged complement attach to the immobolized component of the complex in proportion to their relative concentrations. After incubation for a set time, the fluid sample and reagent are separated. The complex immobilized to the solid phase is then illuminated with radiation of a wavelength chosen to excite fluorescence of the tag, and the fluorescence is measured. The intensity of the fluorescence of the immobilized complex is inversely proportional to the concentration of the untagged complement being assayed.
Alternatively, an assay may be made by immobilizing a quantity of an analog of the moiety to be quantitated (i.e., a substance which is immunologically similarly reactive) and reacting the sample with a known quantity of tagged complement. The tagged complement complexes with both the unknown quantity of the moiety in the sample and the immobilized analog. Again, the intensity of fluorescence of the immobilized complex is inversely proportional to the concentration of the (free) moiety being quantitated.
So-called "sandwich" immunoassays may be performed for multivalent complements to the immobilized component, the attached complement being then further reacted with a tagged analog of the immobilized component. Thus, bivalent antigen may be bound to an immobilized antibody and then reacted with a fluorescent tagged antibody, forming an antibody- antigen-tagged antibody sandwich that may then be separated from the unreacted tagged antibody. The intensity of the fluorescence of thus formed immobilized complex is directly proportional to the concentration of the species being quantitated.
In any of these assays, physical separation of the immobilized complex and the unreacted tagged component is required, and typical prior art methods accomplish this by aspirating, decanting, eluting, or otherwise separating the solid phase and the fluid. Beyond permitting quantitation of the reacted tagged component, such a separation step also reduces the amount of background fluorescence, an important consideration in view of the intrinsic fluorescence of substances which may be anticipated to be present in biological samples (e.g., serum biliriun, tryptophan, various drugs, and the like). Aside from requiring an additional (the separation) step in the laboratory protocol, such procedures determine a reaction end-point, and are therefore not convenient for studies of reaction dynamics.
Beyond actually removing fluid from the solid phase immobilized complex, as is common in separation immunosasays, the separation may be accomplished in situ by restricting fluorescence measurements to the immediate vicinity of the solid phase. Thus, if fluorescence is induced only within molecular dimensions of the surface of the solid phase, only those fluorophores within such a distance, and presumably therefore complexed with the immobilized component, will be excited. A method for inducing and observing fluorescence of a sample at an interface between the sample and another material has been developed by Hirschfeld (U.S. Pat. No. 3,604,927). In this method, the sample is contacted to the face of a prism, and the prism is illuminated with the exciting radiation such that total internal reflection occurs at the face contacting the sample. The sample is thus illuminated by an evanescent wave which penetrates into the sample only a relatively short distance, its electric field amplitude exponentially decreasing with distance from the interface to e.sup.-1 of its surface value typically in less than 1000 Angstroms, the exact effective penetration depth depending upon wavelength, refractive index mismatch, and ray path relative to the critical angle. In this way, a thin surface layer of a sample may be probed. Observations of the fluorescence emitted from this lamina at large angles to the interface effectively filters the exciting radiation from the fluorescent signal.
This procedure has been specifically applied to fluorescent immunoassay by Kronick and Little (U.S. Pat. No. 3,939,350), who teach immobilizing a component of the complex to the prism (or a slide in optical contact with the prism) and observing the fluorescence of those fluorophores excited by the evanescent wave. To the extent that these are fluorophores concentrated by and bound to the immobilized component of the antigen-antibody complex, the method enhances the desired signal relative to the background fluorescence of the sample or reagent. An advantage to this approach is that the intensity of fluorescence may be observed in situ as a function of time, in contrast to separation techniques wherein the solid and liquid phases are separated before measurement, and wherein therefore an endpoint (corresponding to the time at which the separation was effected) is observed. Unfortunately, the technique only isolates a lamina no thinner than several hundred Angstroms, thereby not totally supressing intrinsic fluorescence of the sample nor completely separating bound from unbound reagent. It thus suffers a disadvantage in comparison to the previously described techniques when large intrinsic fluorescence is present or very low titers are to be quantitated.
Further, Kronick and Little's method and apparatus, in common with the wall-coated tubes of prior art fluorescent assays, observes the Lambertian radiation of a fluorescent surface. Efficient utilization of such a source requires a system having a large optical throughput (i.e., a large solid angle--aperture product). Such systems are inherently larger, more complex, and more expensive than systems having smaller throughput. For instance, throughput matching of a radiation detector to such an extended diffuse source requires a large area (and therefore, a more expensive and noisier) detector, generally used in conjunction with large collection optics. The alternative to efficient utilization of the emitted radiation is a loss in system sensitivity.
To compensate for the loss in system sensitivity brought about by poor optical throughput matching of an extended fluorescent source, one may resort to more intense excitation sources and multiple reuse of the excitation radiation by repeatedly reflecting it onto the sample. This, too, is exacerbated by the extent of the area to be illuminated and the need to throughput match the excitation source to the area. Lacking such a match, very intense (and usually complex and expensive) sources are required. Thus, the preferred embodiment of Kronick and Little (U.S. Pat. No. 3,939,350) incorporates a helium-cadmium laser and has a sensitivity sufficient only for the assay of relatively concentrated samples.
An alternative is to more heavily load the tagged component with a (generally already) highly efficient fluorophore. This commonly leads to a degradation in the specificity and avidity of the labelled component for its complement, as well as a reduction in the fluorescence efficiency, which eventually becomes limiting.